Detection of nucleic acids in a sample is useful in diagnostic, therapeutic, forensic, agricultural, food science applications and other areas. Methods of nucleic acid detection include those that use physical separation and detection of a nucleic acid, such as by capturing the nucleic acid in or on a matrix or support and detecting the captured nucleic acids by using a means to visualize the nucleic acid, such as a dye or intercalating agent, or by hybridizing a detectable probe to the nucleic acid. Known methods for separating and detecting nucleic acids use electrophoretic separation of nucleic acids by size, e.g. by using a gel or other chromatographic matrix, followed by staining or attaching a probe to the separated nucleic acids to produce a signal to indicate the presence of the nucleic acid in the sample. Some methods indirectly detect nucleic acids by producing a product made from using a target nucleic acid as a template and detecting the product, e.g., detecting an RNA transcript made from a DNA, or a translated protein made from an RNA transcript. Other indirect methods detect a product made by an enzymatic reaction associated with the nucleic acid to be detected, e.g., an enzyme-linked probe hybridized to the target nucleic acid which produces a detectable response when the enzyme's substrate is provided. Some methods of nucleic acid detection rely on amplifying a nucleic acid sequence to produce a larger quantity of nucleic acid that is detected. Examples of amplification methods include producing many copies of a cloned sequence and in vitro amplification procedures that use enzymatic synthesis of multiple copies of a nucleic acid sequence.
Many of the techniques for detecting nucleic acids require the presence of a relatively large amount or proportion of the target nucleic acid in the sample, while other techniques use nucleic acid amplification to increase the amount or proportion of the nucleic acid to be detected from a smaller amount of the target nucleic acid in a sample. Enrichment of some or all of the nucleic acid present in a sample may facilitate detection of the nucleic acid of interest. Many known procedures for nucleic acid enrichment and detection are laborious, time-consuming, or require use of equipment or hazardous chemicals (e.g., chaotropes, mutagens, or radioactive compounds) that make such procedures undesirable for many applications, such as for rapid screening of many samples, point-of-care diagnostics, or detection at a site outside of a laboratory. Thus, there remains a need for a method that provides relatively simple procedures and sufficient sensitivity and/or specificity to detect a nucleic acid of interest.
The physical nature or relative abundance of some nucleic acids may impede their detection in a sample. For example, small RNA (about 17-27 nt), such as microRNA (miRNA), small or short interfering RNA (siRNA), short hairpin RNA (shRNA), and small nuclear RNA (snRNA) are difficult to separate from other sample components and/or to detect by using known methods. Small RNA are often relatively rare in a biological sample which contributes to the difficulty of their detection. Because small RNA are important regulatory molecules that modulate or silence gene expression via RNA interference (RNAi), they may be important disease preventive or therapeutic agents. Thus, there is a need for a method that rapidly detects the presence of small RNA in biological samples to determine their presence, stability, therapeutic efficacy, or other characteristics in a biological sample without requiring extensive processing or nucleic acid amplification. There is a further need to detect localized small RNA in a variety of biological samples to avoid conditions that lead to inadvertant suppression of non-targeted gene functions by small RNA. Current methods for detecting small RNA or their effects in biological samples are time consuming and laborious, e.g., in situ hybridization, nuclease protection assays, Northern blots to detect RNA, Western blots to detect proteins, immunoassays, and fluorescence detection assays (PCT App. Nos. WO 0044914, Li et al., WO 05 004794, Bumcrot et al.).
This application responds to the need for efficient nucleic acid detection assays by disclosing methods and compositions useful for the rapid detection of nucleic acids in samples, including small RNA in biological samples.